Figure 4A-D. Effect of vimentin ablation on intracellular distribution of RNF26. A. Representative confocal images of parental and vimentin knockout (Vim KO) U2OS cells ectopically expressing GFP-RNF26 (green), fixed and immunostained against endogenous VAP-A (magenta). B. Validation of vimentin knockout. Immunoblots against endogenous vimentin and Vinculin (loading control) of a Vim KO clonal cell line and their parental U2OS cells are shown, representative of three independent experiments. Molecular weight markers are as indicated. C. Quantification of GFP-RNF26 signal distribution expressed as perinuclearity ratio. Graph reports n Parental = 73, n Vim KO = 43 technical replicates from three independent experiments. D. Colocalization (Manders') analysis of VAP-A overlapping with GFP-RNF26 (left plot) and vice versa (right plot). Plots report on subset of cells from data set in (B), same as in (C). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test (D Mann-Whitney U test (C error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.E, F. Effect of vimentin or RNF26 ablation on the intracellular distribution of endolysosomes. E. Representative confocal images of parental, RNF26 KO, vimentin KO #1 U2OS cells, vimentin KO#1 cells ectopically complemented with untagged wild type vimentin (Vim KO + Rescue), fixed and immunostained against endogenous LAMP1. F. Quantification of LAMP1 signal distribution expressed as perinuclearity ratio. Graph reports on n Parental = 145, n R26 KO = 64, n Vim KO#1 = 119, n Vim KO#1/Rescue= 32 technical replicates from three independent experiments.Values capped at 6. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Mann-Whitney U test F error bars indicate median and 95% confidence interval); * p<0.05, ** p<0.01, *** p<0.001.G, H. Effects of vimentin ablation on trafficking of extracellular materials to lysosomes. G. Time-lapse imaging. Representative confocal overlays of parental and vimentin KO#2 U2OS cells, stained with SiR-Lysosome (magenta) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 (green), taken soon after the start (t=5 min) and at the end (t=120 min) of time-lapse are shown. Zoom-ins highlight select PN regions in overlay and single channels. H. Colocalization (Manders') of SiR-Lysosome overlapping SR101 at t=120 minutes following SR101 addition. Graph reports on n Parental = 71, n Vim KO = 69 technical replicates from 3 independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test H error bars indicate mean -/+ SD) ; * p<0.05, ** p<0.01, *** p<0.001.